JPS5931715A - Preparation of carcinostatic substance tf-500 - Google Patents

Abstract

PURPOSE:To obtain the titled novel substance having immune activating action and low side effect, by extracting a mold of bacterium belonging to the genus Fusobacterium with a dialkyl sulfoxide. CONSTITUTION:A mold of a bacterium belonging to the genus Fusobacterium is extracted with a dialkyl sulooxide, a hydrophilic organic solvent (e.g., acetone) is added to the preferably prepared extracted solution to give a precipitate, which is subjected to removal of protein by enzymatic treatment with pronase, and it is subjected to ultrafiltration using a filter membrane having 50,000- 200,000 fraction molecular weight, to give novel carcinostatic substance TF- 500 or its salt having the following properties. Appearance: white gray or light brown powder. Melting point: 180 deg.C (decomposition)-195 deg.C. Solubility: soluble in water, insoluble in methanol, etc. Positive in Molish, phenol sulfate, indole hydrochloride reaction, etc. Elemental analysis (%): C 38-47, H 5-7, N 1-4.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for producing an anti-cancer substance, and more specifically, to an extraction treatment using a dialkyl sulfoxide from bacterial cells belonging to the genus Funbacterium. The present invention relates to a method for producing an anticancer substance TF-500 or a salt thereof having the following properties from a liquid.

(a) White-gray to pale brown powder (b) Has anticancer and immunostimulatory effects (-→ soluble in water, insoluble in methanol, ethanol, acetone, pen 7), chloroform, ethyl acetate, diethyl ether.

2) It shows a clear melting point and begins to decompose at about 180℃,
Decomposes significantly above 195°C.

(e) The infrared absorption spectrum obtained by the KBr tablet method is
3500~3200,2920,2850.1660~
1600.1580~1520.1460~1400°1380~1360.1120~1100.1080~
It has absorption bands near 1000.970 and 840 to 800 on-''.

(f) The ultraviolet absorption spectrum of water bath water with a pH of 7 has strong absorption at the absorption end and absorption in the vicinity of 246 to 280 nm.

()) Molisch reaction, phenol-sulfuric acid reaction, Anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Folin reaction are positive, and nimbidrine reaction is negative.

(Force original analysis value C: 38% to 47% H: 5% to 7% N: 1% to 4%
(S) The sugar content determined by the phenol-sulfuric acid method is approximately 3.
The protein content according to the Lowry-Folin method is approximately 10% or less (in terms of bovine serum albumin).

In recent years, there has been active research into therapeutic methods for treating various cancer patients that enhance the immune function of the host and exert anticancer effects with the help of the immune function.

The present inventors investigated the pharmacological effects of components extracted from bacteria belonging to the genus Humbacterium isolated from the in vitro cavity by dialkyl sulfoxide, and found that certain components were detected. It has anti-cancer properties, and has the effect of increasing host-mediated or host immunity and indirectly exerting anti-cancer effects with the help of immune system. We discovered that it has favorable properties as an anticancer agent, such as being strong and having few side effects, and after conducting various studies on methods for obtaining such a component, we completed the present invention.

The bacteria used in the present invention include anticancer drug 'J [TF-
Any bacterium belonging to the genus Fusonocterium that forms in the 500' region may be used, and a preferred example is Funbacterium nucleatum. Specifically, for example, Fusono (Cterium nucleatum TF-031 (FIRM-
5o7'7, Human TCC-31647) and, as a general knowledge in microbiology, strains with the same properties, ie, natural mutant strains or artificially improved strains, are used.

The mycological properties of Humbacterium nucleatum TF-031 are as follows.

8- (1) Morphology■ Cell shape: spindle-shaped (Fig. 1) ('J) Presence or absence of cell pleomorphism: None■ Presence or absence of motility: None■ Presence or absence of spores: None■ Gram stain nigerum negative■ Anti-acidity: Negative (2) Growth status in medium ■ TF-a agar plate and slant medium External shape: Two circles Size: Approximately 1 mm Raised two-handed spherical structure 8M droplet-like surface: Smooth Edge: Smooth Color: Milky white Transparency: Opaque (Tp-a liquid medium Growth level: Strong turbidity: Coagulum precipitation: None Surface growth: Clear, no growth in approximately 5 mm trenches Gas: None; 3) Physiological level ■ Production of hydrogen sulfide 2 10 (reduction of nitrate ■ Formation of butyric acid 20 ■ Formation of indole 20 ■ Urea zeny ■ Catalan zeny 11- ■ Hydrolysis of starch (8) Attitude toward oxygen: Anaerobic ψ Formation of ammonia 20 qO Production of carbon dioxide 20Q0 Growth range: PH5-8.5 Temperature 30-45°C (3,2) Production of gas from sugar L-7 Ravi/-ose (H), D-xylose (H), J〕
- Glucose 8% D-Mannose ←), D-Fructose H, D-Galactose H1 Maltose (Ha), Sucrose (Ha), Trehalose (Ha), Nruvit (Ha), Mannitol (Ha), Inosit (Ha), Percy's -Nual-Glycerin H1 Starch (C)
When examined in the light of Determinative Bacteriology 12-8th Edition, this strain is Fusobacterium nucnoatum (Fusobacterium tar-ium).
nuclaatum) and belongs to this group.

Next, an example of the method for producing the anticancer substance TF-500 of the present invention will be schematically explained as follows.

Margin below Collection of both components from bacterial cells that have anticancer effects Belongs to the genus Humbacterium Culture solution containing a plane filtration scoop body extract liquid Deproteinization treatment and/or ultrafiltration anticancer substance F-500 The above manufacturing method is specifically shown below.

(a) Culture Culture of the bacteria belonging to Hubaczolium glume is carried out by a conventional method for culturing anaerobic bacteria.

Namely, nitrogen sources such as cow brain, heart extract, and various peptones; vitamin sources such as yeast extract; inorganic salts such as sodium chloride; carbon sources such as nigercose and lactose; A medium containing a reducing agent such as sodium or thioglycolate is purified with sodium hydroxide.
Adjust to H6~8.5, preferably 6.5~7.5, inoculate the bacteria, and heat under anaerobic conditions at 30~42℃, preferably 32℃.
Allow to stand or culture with stirring at ~37°C for 1 to 5 days, preferably 1 to 4 days.

15- In particular, it is wise to use the medium (hereinafter referred to as TF medium) listed in the ingredient table below. However, as a nitrogen source, Plain Heart Infusi 1 of Horse Brain and Heart Extract
yii It is not necessary, but you can substitute heart infusion which is a cow's heart extract, beef extract, fish meat extract, dried corns diblica, etc. Of the various peptones, proteose peptone and phyton peptone are not necessarily necessary, and tryptocase peptone can be substituted with polybefton.

Note that when agar is not used, it is preferable to perform agitation culture.

1] (i- (b) Culture - Collection of bacterial cells from nutrient solution The supernatant liquid is removed from the culture liquid obtained above to obtain bacterial cells. The supernatant liquid can be removed by conventional methods such as centrifugation and Celite. The Okisu method using Hironoki Soybean can be used.The bacterial cells obtained in this way can be washed with physiological saline and then dried by a normal method such as freeze-drying, or washed with physiological saline. After washing, the cells can be washed in a short time with a hydrophilic organic solvent such as ethanol or acetone, and then dried cells can be obtained by a conventional drying method such as air drying.

(,) For example, 10 to 50 times the amount (volume/
A dialkylsulfoxide such as dimethyl sulfoxide or diethyl sulfoxide (weight ratio), preferably dimethyl sulfoxide, is added and stirred at room temperature to 80°C for 30 minutes to several days, preferably around 65°C for 3 to 5 hours. Perform an extraction operation. Next, insoluble materials are removed by conventional means such as decantation, centrifugation, and filtration, and the resulting extract is mixed with a hydrophilic organic solvent, such as ketones such as acetone, diethyl ketone, and methyl ethyl ketone, as well as methanol and ethanol. ,
Alcohol such as Inglovir alcohol is added at low temperature at least 5 times the amount (volume!-/volume ratio) to the extract.

The hydrophilic organic solvent is preferably added under acidic conditions, for example, a hydrochloric acid acidic acetone solution is added and left at around 4° C. for several hours to several days. Separate and collect the precipitate using a conventional method. The precipitate thus obtained is further subjected to protein removal treatment or ultrafiltration, either alone or in combination.

In addition, when the bacterial cells are collected by a method such as washing with physiological saline and freeze-drying, the anti-cancer substance TF-500 or its salt may be obtained by omitting the protein removal treatment or ultrafiltration operation.

(al Protein removal treatment, ultrafiltration, and target product collection step) The precipitate obtained in (c) above is subjected to ultrafiltration after protein removal treatment, or protein removal treatment or ultrafiltration is performed.V
Accordingly, the anticancer substance TF-500 or a salt thereof can be produced.

The protein removal treatment referred to herein may be any treatment known in the art, and when subjecting cicadas to enzyme treatment for protein 20-leukolytic enzyme, the precipitate obtained as described above is first treated. What is necessary is to dissolve or suspend the substance in water or a buffer solution, add a proteolytic enzyme, and perform enzyme treatment according to a conventional method.

Examples of the protease include pronase, papain, trypsin and chymotrypsin, preferably pronase and trypsin. In or during the enzyme treatment, it is preferable to adjust and maintain the pH of the aqueous solution at 7 to 8, and for this purpose, sodium hydroxide, potassium hydroxide, soda carbonate, and sodium carbonate are used. During enzyme treatment, a small amount of organic solvent such as chloroform or toluene may be added to prevent spoilage of the reaction solution.
It is desirable to add t. The amount of enzyme used is usually about 1 to 2% of the powder (solid) to be subjected to enzyme treatment.
Sweetness around (weight ratio) is used.

The above enzyme treatment is usually carried out at 30 to 40°C for 1 to 72 hours, preferably 24 to 48 hours.

Also, for example, add about 1% enzyme (X amount ratio) to the powder (solid), perform enzyme treatment for 1 to 24 hours, and then add about 1%
Enzyme treatment may be performed by adding ('N amount ratio) enzyme.

After the above protein removal treatment, the anticancer iMTF is extracted from the water-soluble portion from which insoluble matter is removed by operations such as centrifugation and filtration, if desired.
-500 fractions are collected. From this water-soluble part, TF-5
To collect both parts of 00, for example, a division method based on pH,
After processing by combining one or more of a precipitation fractionation method using a hydrophilic organic solvent and a separation method using an ion exchanger, the product may be subjected to ultrafiltration (these operations may be repeated twice). To give a specific example of the collection method, the water-soluble part is preferably adjusted to pH 2.5 or less, and trichloroacetic acid may be added if desired). If a precipitate is formed, it is removed, Add a hydrophilic organic solvent, such as alcohol, to the dissolving part at a rate of 30 to 80% (volume ratio), and use a favorable mechanism of 60 to 8
0% (volume ratio), collect the precipitate of the resulting fraction of the target product, and if desired, then add this precipitate to
For example, Dowex Type 1 (product name), Amberlight I
Treat with a strong basic anion exchanger such as RA-400 (trade name) (this operation may be repeated several times).
= 50,000 to 200,000), shaken and desalted, and dried to obtain anticancer substance TF-500.

This TF-500 is 0.2% (Xi/vol.i) in water.
It becomes a clear solution.

Instead of the above-described operation of performing ultrafiltration after protein removal treatment, each of the protein removal treatment and ultrafiltration described in section n above can be performed independently.

The anticancer substance TF-500 obtained as above is
It may be prepared as a pharmaceutically acceptable non-natural salt according to a conventional method.

Specific examples include alkali 4[salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, and the like.

24 - The pharmacological action of the anticancer substance TF-500 of the present invention is as follows.

(1) Effect of inhibiting colony formation of cancer cells
, 25, 698 (1965)), He1a8-
After culturing 3 cells in Eagles MEM medium supplemented with 10% bovine serum for 3 to 5 days, the culture medium was removed and the cells were cultured in PB containing 0.01% ethylenediaminetetraacetic acid and 0.1% trypsin.
B (to) solution (8.Of sodium chloride, 0.2f of potassium chloride in IJ.

Sodium hydrogen phosphate 1.15f and dihydrogen phosphate + Jum 0.2f 'f: Aqueous solution containing]
The cells are peeled off from the glass surface of the culture bottle and diluted with Eagles MKM medium supplemented with 20% bovine serum, so that each diluted solution contains 300 cells. This cell suspension was heated for 2 hours at 37°C in a 002 incubator.
Anticancer substance TF-500k250.5 after 4 hours of culture
0.00.Add to the culture solution at 1000 μt/passage.

Two experiments were conducted, one in which serum was added (20%) to the culture medium and one in which serum was not added. After 24 hours of treatment, cells were washed with serum-free culture medium, culture medium containing 2° flyer serum was added, and CO! Culture in an incubator for 6 days. After culture, cells were fixed with ethanol, G1θmsa
The cell viability was determined by staining and counting the number of colonies. The results are shown in Table-1. Similarly, the survival rate of cells in Tatsunaka was calculated using the following formula.

Table 1 “Medium” and “culture solution” used to investigate this effect
Both are Eagles MEM medium and culture solution.

) Immunostimulatory effect Using 3 ICR mice per group, the anticancer substance TF-5
5μ for 00? ,50μ? and 500 μr/Kpe was administered intraperitoneally. Perikan Dra 24 hours after administration
wing Ink 17 Elack
Carbon suspension prepared by mixing Wagner's PlLm, 13 and 2mef of physiological saline containing 3% gelatin 0
．． Injected from the tail vein of 27dk mice [-1 after injection 1.5.
After 10 and 15 minutes, blood o, o2dTh was collected from the orbit into a heparin-coated tube using Madcrit capillary sound, immediately diluted with 1.6 ml of 0.1% sodium carbonate aqueous solution, hemolyzed, and compared by color at a wavelength of 675 nm to determine whether the blood was phagocytic. Coefficient (Pbagocytotic 1ndex): K
The value f was determined by the formula of Halpern et al. In addition, to the control group, 0.2 liters of physiological saline was administered from 1 to 1.

t　　　　-t.

(The amount of charcoal in the blood when co = to, the amount of charcoal in the blood when C=) The results are shown in Table-2.

27- Table-2 The rice obtained in Example 1 was used.

(3) Antitumor effect IO against Sarcoma 180 cancer cells
Sarcoma 180 g 5 cells (1 x 10 cells per mouse) were subcutaneously transplanted into the lower axilla of R mice (female, 6 weeks old).

Next, the test substance was dissolved in a 5% glucose aqueous solution, and 0.2 TILt of the solution was intravenously administered on the 10th and 12th day after cancer cell transplantation. In addition, to the control group, 5% glucose aqueous solution 0.2/1 gold was administered in the same manner. The results are shown in Zenko-3.

28- Clothes -3 The rice obtained in Example 1 was used.

In Example 2, I 〃 3, T in acutely toxic mice (ICR strain, female, 6 weeks old: 1v administration)
? -500 L horses. The value was greater than 10 kg/Ky.

As is clear from the above pharmacological experiments, the anticancer substance TF-500 obtained by the method of the present invention is useful as an anticancer agent, and is expected to be effective when used for various cancer diseases.

The anticancer substance TF-500 can be used as it is or in the form of a non-toxic salt in the form of oral, injection, crude drug, etc. by conventional methods. Oral preparations may contain various excipients and may be in the form of capsules, tablets, powders, or granules. The injection may be subcutaneous, intramuscular, or intravenous, and may be in the form of a suspension, solution, or powder to be dissolved before use. Also, injections and (~
For example, the above-mentioned powder can be dissolved or suspended in physiological saline, an aqueous glucose solution, or an aqueous solution containing a local anesthetic.

When the anticancer substance TF'-500 and its non-toxic salts are used in patients, the dosage is appropriately selected depending on the patient's symptoms, but in general, for adults it is 0.00.
It is preferable to administer K9 once to several times a day, and administration methods include oral, subcutaneous, intramuscular,
Preferably, it is administered intravenously or by injection into the affected area.

Next, the present invention will be explained with reference to Examples and examples of sheathing materials.

Example 1 (1) 9oI! Tryptocase Peptone 1190F, Heart Infusion 1050F, (-St Extract 2101F) to 70 J of distilled water in a jar fermenter (Marubishi Rika Institute pMBJ-U type).
, salt 525 F, glucose 8401, lactose 7
00f, T'F-f medium containing 71 t of sodium sulfite, 35 t of thioglyconautium and 175 t of dibasic potassium phosphate and sterilized for 15 minutes at 118°C. After cooling, add nitrogen gas to the culture solution at 2zsomJ/min.
Vent for hours.

Humbacterium nucleatum TF-031 (F RM-50), which had been precultured in the TF-f medium described above
77, ATOO-316471 preculture solution approximately 900 m
1 is inoculated into the above-mentioned 1-culture solution.

Cultivation was carried out at approximately 33°C with total nitrogen gas inflow (25 omJ/+) i
Stirring (90 r, p, m) for 4 days.

(2) The culture solution obtained above was subjected to ISM'ti centrifugation (4X1 (1" rpm 10 minutes) to collect bacterial cells,
1 part saline for bacterial cells! The dried bacterial cells were washed with water, then degreased with 11 parts of ethanol and 11 parts of acetone, and dried.
I got f. The bacterial cells were suspended in 300 g of dimethyl sulfoxide and extracted with stirring for 4 hours while heating to 65°C. Next, liquid F was separated by suction filtration, and acetone 2 was added to it.
After adding E and concentrated hydrochloric acid 81, the mixture was allowed to stand at 4° C. for 2 days to precipitate and ripen.

This precipitate was collected by centrifugation (4 x 10"rpm, 10 minutes) (748 cm).

(3) Precipitate 748111F'i water 15 obtained above
1 and pH 7.8 with ammonium carbonate aqueous solution.
Adjust to ~8.0. In addition, pronase E (Kaken Kagaku Dan product name: 1,000,000 tyrosine units/xw
r and 5 my were added twice every 30 minutes, several drops of toluene were added, and enzyme treatment was performed at 37° C. for 24 hours. After adjusting the pH to 1 by adding hydrochloric acid to the treatment solution, ethanol was added to a final concentration of 80%, and centrifugation (4X 10
Collect the precipitate that precipitates at 10 minutes (rpm, 10 minutes).
288.6■).

(4) 50% water to 288.6'a9 of the precipitate obtained above
1 and ultrafilter [TK-50 (Toyo F Paper Co., Ltd. Tai Ultrafiltration Membrane brand name 2 cutoff molecular weight 5X10
Concentrate 100 times by ultrafiltration using 'l. This concentrated solution is filtered through a Millipore filter (trade name, manufactured by Nippon Millipore Limited) with a total pore diameter of 0.2 μm, and then freeze-dried to obtain 264IIg of a freeze-dried product of the anticancer substance TF-500.

The anticancer substance TP-500 thus obtained had all the properties described above, and its infrared absorption spectrum and ultraviolet absorption spectrum -35= were as shown in FIGS. 2 and 3.

Example 2 (1) Example 1 - The culture solution of 701 obtained in (1) was fractionated by filtration using i-tone Celite (Hyfloss Parcel, a product manufactured by Johns Manville, USA). The mixture of bacterial cells and celite was washed with 20 A' of physiological saline, and then degreased and washed with ethanol 31 and acetone 3j in order, resulting in a mixture of 3.2 kg of bacterial cells and celite.
(dry weight) was obtained. This mixture was suspended in 320 f total dimethyl sulfoxide 4001, and extracted with stirring for 4 hours while heating to 65°C. Then, by suction filtration, p
Separate the liquid gold and add 2.5j of acetone and concentrated hydrochloric acid IQ to it.
After adding ag, the mixture was allowed to stand at 4° C. for 2 days to precipitate and ripen. This precipitate was centrifuged (4 x 10” rp
m, 10 minutes) (94 ql.

(2) The precipitate obtained above was dissolved in 94 qft of water and 100 times ultrafiltered using Ultrafilter UK-50 (Toyo F Paper Co., Ltd. ultrapure membrane trade name: molecular weight cutoff 5X10JK). Concentrate into.

This concentrated solution was filtered through a Millipore filter (trade name of Japan Millipore Limited) with a pore size of 0.2 μm, and then freeze-dried to produce a freeze-dried product of the anticancer substance TF-500.
2. Obtained at 6Q'.

The anticancer substance TF-5(]Ot′ obtained in this way
It had the properties described above, and its infrared absorption spectrum was as shown in FIGS. 4 and 5.

Formulation Example 1 pH 7 containing the anticancer substance TF-500 obtained in Example 1,
Fill all vials with an aqueous solution of 0 to 7.5 to obtain 0.5 or 1 unit of freeze-dried anticancer substance 'rF-500. When using this, sterile physiological saline, lidocaine 0.5%
The containing liquid is dissolved isobarically in a 5% aqueous glucose solution and used as an injection solution.

Formulation Example 2 pH 7 contained in the anticancer substance TP-500' obtained in Example 2
, 0 to 7.5 is filled into a vial to obtain 065 or 1 unit of lyophilized anticancer substance TF-500.

When used, it is used as an injection solution by dissolving it in sterile physiological saline, a solution containing 0.5% lidocaine, or an aqueous solution of pentate glucose.

[Brief explanation of drawings]

Fig. 1 is a micrograph showing the morphology of Funbacterium nucleatum TF-031 used in the present invention, Fig. 2 is an infrared absorption spectrum of the anticancer substance TF-500 obtained in Example 1, and Fig. 3 is the ultraviolet absorption spectrum of the same substance.
Figure 5 shows the infrared absorption spectrum of the same substance. 39-123- Continued from page 1 0 Inventor 9-1 Matsuwaka-cho, Minamimukai Toyama City 0 Inventor Takako Hori Prefectural Housing 4-02, 1383 Mukaishinjo, Toyama City

Claims (1)

[Scope of Claims] (1) The cells of fungi belonging to the genus Humbacterium are subjected to extraction treatment with gold dialkyl sulfoxide, and the anticancer substance TF-500 or its salt having the following properties is extracted from the resulting extract. Anticancer substance TF-
500 and its salt manufacturing method. (a) White-gray to light brown powder (b) Has anticancer and immunostimulatory effects. 0 → Dissolves in water vc-a, insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, and ethyl ether. ) It does not show any clear melting point and begins to decompose at about 180℃,
Decomposes significantly above 195°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3500~3200,2920,2850.1660~
1600.1580~1520.1460~1400°1380~1360.1120~1100.1080~
It has absorption bands near 1000.970 and 840-800 cm-'. (f) The ultraviolet absorption spectrum of an aqueous solution at pH 7 has strong absorption at the absorption end and absorption near 246 to 280 nm. (G) Molisch reaction, phenol K acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Folin reaction are positive, and ninhydrin reaction is negative. (Power elemental analysis value: C: 38% to 47% H: 5% to 7% N = 1% to 4% (su) Sugar content by phenol-sulfuric acid method is approximately 30% to
12% (in terms of glucose) and the protein content by the tetrauri-folin method is about 10% or less (in terms of bovine serum albumin n). (2) Cells of fungi belonging to the genus Humbacterium were subjected to extraction treatment with Schiff sulfoxide, and a hydrophilic organic solvent was added to the resulting extract, and the resulting precipitate was subjected to protein removal treatment. 2. The manufacturing method according to claim 1, further comprising ultrafiltration. (3) The preparation according to claim 2, wherein the dialkyl sulfoxide is dimethyl sulfoxide. (4) The production method according to claim 2 or 3, wherein the hydrophilic organic solvent is acetone. (5) The production method according to any one of claims 2 to 4, wherein the protein removal treatment is an enzyme treatment using a protease. (6) The production method according to claim 5, wherein the enzymatic treatment with a proteolytic enzyme is pronase treatment. (7) A patent claim characterized in that the microbial cells of the 7th bacterium genus are subjected to extraction treatment with dialkyl sulfoxide, a hydrophilic organic solvent is added to the resulting extract, and the resulting precipitate is subjected to ultrafiltration. The manufacturing method according to item 1. (8) The manufacturing method according to claim 7, wherein the dialkyl sulfoxide is dimethyl sulfoxide. (9) The manufacturing method according to claim 7 or 8, wherein the hydrophilic organic solvent is acetone. α1 The production method according to any one of claims 2 to 9, in which ultrapolar filtration is carried out using a filtration membrane with a molecular fraction of 1ii50,000 to 200,000.(II) Belongs to the genus Hubbacterium 11. The production method according to any one of claims 1 to 10, wherein the bacterial cells are Funbacterium nucleatum.